Congener independent detection of microcystin and nodularin congeners

ABSTRACT

The present invention relates to a protienaceous compound or functionally active derivative or part thereof having a binding site for a group represented by formula (I) which is part of a group of toxins derived from various cyanobacteria, to a method for its production, to diagnostic kits and to an affinty matrix (e.g. for use in immunoaffinity columns, online detection and purifications devices) containing the proteinaceous compound as well as to methods for substantially decreasing the amount of a compound containing the group represented by formula (I) in fluids or for concentrating compounds, e.g. toxins, containing the group represented by formula (I) from fluids such as crude water samples, extracts of algae or other tissue samples, e.g. to determine toxin concentrations.

The present invention relates to a proteinaceous compound orfunctionally active derivative or part thereof having a binding site fora group represented by the following formula (I)

which is part of a group of toxins derived from various cyanobacteria,to a method for its production, to a diagnostic kits and to an affinitymatrix (e.g. for use in immunoaffinity columns, online detection andpurification devices) containing the proteinaceous compound as well asto methods for substantially decreasing the amount of a compoundcontaining the group represented by formula (I) in fluids or forconcentrating compounds, e.g. toxins, containing the group representedby formula (I) from fluids such as crude water samples, extracts ofalgae or other tissue samples, e.g. to determine toxin concentrations.

Due to increasing settlement, industrialisation and intensiveagriculture wide spread problems of water pollution have arisen. Thiswater pollution and the following eutrophication has led in many casesto the development of blooms of blue-green algae (i.e. cyanobacteria).The environmental factors which are responsible for the development ofsuch blooms of cyanobacteria are up to now almost unknown. In general,blooms of cyanobacteria can be found in eutrophic bodies of water, e.g.under such conditions as relatively high nutrient levels (phosphate andnitrate), water temperatures of between 15 to 30° C. and pH-values ofbetween 6 and 9 or higher (Wicks et al., 1990).

A severe problem of the development of blooms of cyanobacteria is thatcyanobacteria produce a broad variety of toxic substances. Accordingly,since the end of the last century there has been an increasing number ofcases of intoxication and even deaths of humans, animals, especiallybirds and fishes, which could be demonstrated to be caused by the use ofwater which was contaminated with cyanobacteria after chlorination andfiltration for medical purposes (cases of deaths in the dialysis centersof Caruaru, Brazil, 1996 and Evora, Portugal, 1995), by the consumptionof contaminated drinking water or even of clumps of cyanobacteriathemselves (Francis, 1878; Falconer et al., 1983; Carmichael, 1984;Beasley et al., 1989; Mahmod et al., 1988; Skolberg et al. 1984).

The toxin producing cyanobacteria can be subdivided into species whichsynthesize mostly hepatotoxic peptides such as Microcystis sp., Nodularasp. and Oszillatoria sp., and other genus which produce mostlyneurotoxic alkaloids such as Anabaena and Aphanizomenon (Carmichael etal., 1990). Studies of different strains of M.aeruginosa revealed that,depending on strain and habitat, the cyanobacteria produce differentcongeners and amounts of a toxin (Sivonen et al., 1992 a–c).

Cyanobacteria can secrete the intracellularly produced toxins into thesurrounding water (Watanabe et al., 1992 a,b). Further studies showedthat the microcystin congener microcystin-LR is photostable, however, itcan be microbially degraded (Watanabe et al., 1992 a; Tsuji et al.,1994; Cousins et al., 1996). Under aerobic conditions and in culturemedia which were inoculated with bacteria, the halflifetimes ofmicrocystin-LR and -YR were more than 45 days (Watanabe et al., 1992 a).In contrast, half-lifetimes of less than 5 days were determined inseawater (Cousins et al., 1996). Under unfavorable conditions (i.e. coldtemperatures and minimal presence of specific populations of microbes)microcystins may persist several days to even months and, therefore, mayrepresent a potential danger for humans via the drinking water supply.

Accordingly, the increased incidence of gastroenteritis and livercarcinomas in humans has been attributed to the consumption of drinkingwater which was contaminated with cyanobacterial hepatoxins (inparticular microcystin-LR) in several studies, although a directrelation between chronic microcystin-LR exposure and the development ofliver carcinomas has not yet been proven (Tisdale, 1931; Keleti et al.,1981; Billings, 1981). Clinical indications of microcystin toxicoses inmammals is characterized by weakness, anorexia, mucous pallor, muscletermor, forced expirations and death by hypovolemic shock which iscaused by intrahepatic hemorrhagia and/or liver failure (Theiss et al.,1988; Jackson et al., 1984).

Mammals seem to take up microcystin orally, and the toxin reaches theliver with the blood via a highly specific transporter mechanism(organic anion carrier) (Eriksson et al, 1990; Hooser et al., 1990;Runnegar et al., 1991). One molecular mechanism of the serious effectsof microcystin seems to be its binding to the catalytic subunit ofproteinphosphatases 1 and 2A which leads to their inhibition (Erikssonet al., 1990; Yoshezawa et al., 1990; Matsushima et al., 1990; Honkanenet al., 1990; McKintosh et al., 1990; McKintosh et al., 1995; Runnegaret al., 1996). After accute intoxication of high microcystinconcentrations, the inhibition of proteinphosphatases leads tohyperphosphorylation of intermediate filaments which, in turn, isfollowed by collapse of the cytoskeleton, loss of the cells' structure,extensive intrahepatic hemorrhage and necrosis of the hepatocytes(Eriksson et al., 1990; Falconer et al., 1981, 1992). Similar to otherproteinphosphatase inhibitors (e.g. calyculin-A, okadaic acid), thechronic exposure of mice to microcystin-LR leads to promotion of livertumors (Falconer, 1991; Nishiwaki-Matsushima et al., 1992).

Due to the high toxicity and carcinogenicity of hepatotoxiccyanobacteria toxins and the potential chronic exposure of organisms(humans as well as animals) to these toxins via the drinking water thereis an urgent need to detect toxic blooms of cyanobacteria early and todecrease the concentration of cyanobacteria toxins in drinking water.

Since it has been difficult to analytically and routinely detect thedifferent microcystin and nodularin congeners with the requiredsensitivity (Kenefick et al., 1993; Lawton et al., 1994), prior artstudies have concentrated on the destruction of the cyanobacteria toxinsduring the drinking water purification process. Mostly, continuousmethods have been studied which can be carried out under routineconditions such as sand filtration, binding to activated carbon anddestruction by chlorination (James et al., 1994). However, these studiesrevealed that neither sand filtration nor chlorination, UV-irradiation,treatment with hydrogen peroxide or potassium permanganate norfiltration via activated carbon could substantially remove thecyanobacteria toxins from drinking water. In this case a further problemseems to be the treatment of the raw water which is contaminated withcyanobacteria. The chlorination or the treatment of the cyanobacteriawith copper sulfate leads to the release of the cyanobacteria toxinswhich are present in the cytosol without destroying the toxins to eventhe lowest degree. Also, the chlorination of sand filtered water isineffective. Only the filtration via activated carbon seems to beappropriate to remove a considerable amount (about 60% to 80%) of thetoxins. However, this purification performance was only reached for alimited period of time due to a relatively quick saturation of theactivated carbon particles. Therefore, after about 10,000 bed volumes (1bed volume=volume of the activated carbon) the filters became leaky.

Therefore, the technical problem underlying the present invention is toprovide a novel system for the reliable detection as well as the removalof all kinds of hepatotoxic cyanobacteria toxins such as microcystin andnodularin congeners, particularly in and from, drinking water and othersources.

The solution to the above problem is provided by the embodiments of thepresent invention as characterized in the claims.

In particular, the present invention relates to a proteinaceous compoundor functionally active derivative or part thereof having a binding sitefor a group represented by the following formula (I)

which is part of a toxin derived from a cyanobacterium, wherein group R¹represents a halogen atom, preferably Br, —OSO₃, —OR′ or —NR′₂ group R²represents hydrogen, (C₁–C₄)alkyl, (C₁–C₄)alkoxy, (C₁–C₄)acyl,(C₁–C₄)aminoacyl or (C₁–C₄)carboxaminoacyl, or the groups R¹ and R² areconnected to each other to form a cyclic compound, the groups R³ whichmay be the same or different are each independently selected from thegroup consisting of hydrogen and (C₁–C₄)alkyl,group R⁴ represents(C₁–C₄)alkoxy, and wherein the phenyl group may be substituted orunsubstituted.

The term “proteinaceous compound or functionally active derivative orpart thereof” means a compound which is capable of binding theabove-described group of formula (I) and substantially consists of oneor more polypeptides. The functionally active form of the proteinaceouscompound according to the present invention may be a monomeric or ahomo- or heterodimeric, -trimeric, -tetrameric or other oligomeric form.

The term “binding site” for the group as defined above means athree-dimensional arrangement of atoms of the above proteinaceouscompound which is able to specifically interact with the group offormula (I) as defined above. The specific interaction may be any kindof chemical and/or physical interaction and comprises covalent binding,electrostatic interactions, hydrogen bonding, Van-der-Waals- as well ashydrophobic interactions.

Preferably, the group R¹ in the formula (I) represents independentlyfrom each other hydrogen, substituted or unsubstituted (C₁–C₄)alkyl or(C₁–C₄)acyl, when bound to nitrogen. According to a further preferredembodiment of the proteinaceous compound as defined above, the groups R³in the above formula (I) each represent methyl and group R⁴ representsmethoxy.

According to a further preferred embodiment of the proteinaceouscompound of the present invention, group R¹ represents acylamino andgroup R² represents (C₁–C₄)acyl, or group R¹ represents glycyl orD-alanyl, respectively, and group R² represents acetyl, or group R¹represents -NH₂ and group R² represents glutamidyl or2-aminoproprionamidyl, respectively.

Preferably, the group represented by the above formula (I) is part of atoxin selected from the group consisting of microcystin and nodularincongeners. Microcystin (MC) and nodularin congeners are hereinafterreferred to as microcystin-XY and nodularin-XY.

The chemical structures of M. aeruginosa and Nodulaia sp.-hepatotoxins(i.e. microcystin-XY and nodularin-XY) are described in several priorart studies (Botes et al., 1982 a, d, 1994, 1985; Rinehard et al.,1988). Microcystin-XY and nodularin-XY are cyclic peptides consisting ofseven or five, respectively, amino acids. The following formularepresents microcystin-LR.

Nodularin-XY and microcystin-XY share the same specific characteristicamino acid (ADDA). The basic structure of microcystin-XY congenersconsists of five non-variable amino acids: β-methylasparaginic acid,alanine, N-methyldehydroalanine, glutamate, and3-amino-9-methoxy-2,6,8-trimethyl-1phenyldeca-4,6-dienic acid (ADDA).The differences between individual microcystin congeners are based onthe two variable L-amino acids which are, for example, L-arginine andL-leucine in microcystin-LR and two times L-arginine in microcystin-RR,respectively. Normally, cyanobacteria produce a mixture of differentforms of the toxins. The isolation of microcystin-XY from natural bloomsof blue-green-algae resulted in up to six different microcystincongeners, and toxin concentrations up to 10 mg per 9 of dry mass ofalgae were determined (Wicks et al., 1990; Tsuji et al., 1994; Tencallaret al., 1994, 1995).

An especially preferred example of the proteinaceous compound accordingto the present invention is a polyclonal, monoclonal or recombinantantibody or a functionally active derivative or fragment thereof. Therecombinant antibody may be produced by the translation and expressionof any part of the genes coding for polyclonal or monoclonal antibodiesand/or selection by screening of a phage display library using the grouprepresent by the above formula (I).

The proteinaceous compound according to the present invention, e.g. apolyclonal, monoclonal or recombinant antibody or functionally activederivative or fragment thereof, has the advantage to be capable ofbinding to all congeners of the cyanobacterial hepatotoxins, e.g.microcystin and nodularin congeners which contain as a part of theirstructure the ADDA moiety.

In contrast to the proteinaceous compound of the present invention, thecommercially available antibodies or ELISA kits, respectivley, are onlycapable of recognizing a very limited number of microcystin congeners.This means that the toxicity of blooms of cyanobacteria can be massivelyunderdetermined by the use of the antibodies or kits, respectively,known so far.

A further embodiment of the present invention relates to a method forthe preparation of the proteinaceous compound as defined above,comprising the steps of

-   -   (a) preparing a compound containing a group represented by the        formula (I) as defined above and    -   (b) coupling the compound of step (a) to a carrier.

The “carrier” is not particularly limited to a specific embodiment andmay be, e.g. any polymeric substance. For example, carriers which aresuitable for the above method may be selected from the group consistingof polyethyleneglycol, proteins, polypeptides, polysaccharides and solidphase supports such as plastic supports. Preferably, the protein carrieris selected from bovine serum albumin (BSA), ovalbumin (OVA) cationisedbovine serum albumin (cBSA), and horseradish peroxidase (HRP).

In another preferred embodiment of the present invention, the abovemethod further comprises the steps of

-   -   (c) immunizing an animal with the conjugate obtained in step (b)        above and    -   (d) isolating the animal's blood, blood serum and/or        spleenocytes.

In a further preferred embodiment, the above method further comprisesthe steps of preparing antisera from the animal's blood serum obtainedin the above step (d) for the preparation of polyclonal antibodies.According to another preferred embodiment, the method of the presentinvention further comprises the steps of preparing monoclonalantibody-producing hybridoma cells from the animal's spleenocytesobtained in the above step (d). Yet another preferred embodiment of theabove-defined method comprises the further steps of preparingrecombinant antibodies including the isolation of the genetic material(DNA) from cells present in the animal's blood or fromantibody-producing hybridoma cells.

A further embodiment of the present invention relates to a diagnostickit containing the proteinaceous compound as defined above.

Another embodiment of the present invention relates to an affinitymatrix containing the proteinaceous compound as defined above coupled toa polymeric resin.

The proteinaceous compound according to the present invention, e.g. apolyclonal, monoclonal or recombinant antibody or a functionally activederivative or fragment thereof as defined above, is particularly usefulfor the detection of a compound containing the group represented by theabove formula (I), for concentrating the toxins from crude extractsprior to analysis to determine toxin concentrations as well as tosubstantially decrease the amount of a compound containing the grouprepresented by the formula (I) in a fluid, pharmaceutical or foodpreparation.

Therefore, a further embodiment of the present invention relates to amethod for concentrating a compound containing the group represented bythe formula (I), e.g. a toxin, from a fluid such as crude water samples,extracts of algae or other tissue samples, or for substantiallydecreasing the amount of a compound containing the group represented bythe formula (I) in a fluid, e.g. water such as hemodialysis water,drinking water or water derived from rivers, lakes and oceans,comprising the steps of

-   -   (a) preparing the proteinaceous compound as defined above,    -   (b) coupling the compound obtained in step (a) to a polymeric        matrix, and    -   (c) contacting the fluid with the polymeric matrix obtained in        step (b).

Furthermore, the above method may also be applied to the cleaning of anyother sources of cyanobacteria toxins such as, for example, food stuffs.

The Figures show:

FIG. 1 is a diagram showing a flow chart for the strategy of preparationof an anti-ADDA antibody according to the present invention.

FIG. 2 is a diagram showing preferred strategies for the coupling of anADDA-hapten to a protein.

FIG. 3 shows several ADDA-derivatives which were synthesized for theproduction of the antibody useful for congener independent detection ofmicrocystin and nodularin congeners.

FIG. 4 is a diagram showing the general principle of the indirectcompetitive microcystin enzyme-linked immunosorbent assay (MC-ELISA).

FIG. 5 is a diagram showing the crossreactivity with respect todifferent microcystin congeners (MC-LR, -RR and -YR) and nodularin ofthe ant-ADDA antibody (ADDA-824new, i.e. 26/06/00) raised in sheep whichis directed against ADDA-HG coupled to ovalbumin.

FIG. 6 is a diagram showing the direct ELISA method and its sensitivityfor detection of MC-YR. The anti-ADDA antibody(ADDA-825-^(bleed,14/12/98)) was raised in sheep and directed againstADDA-HG coupled to ovalbumin.

FIG. 7 is a diagram showing the indirect ELISA method using a monoclonalantibody and ist sensitivity for detection of MC-YR. The anti-ADDAantibody (ADDA-3G10B10) was raised in mice and directed against ADDA-HGcoupled to ovalbumin.

The present invention is further illustrated by the followingnon-limiting examples.

EXAMPLE ADDA Hapten Synthesis

The starting material N-Boc-ADDA-Me (35) was prepared by the publishedroute: Humphrey, J. M.; Aggen, J.; Chamberlin, A. R. J. Am Chem. Soc.1996, 118, 11759–11770. “Synthesis of the Serine-threonine PhosphataseInhibitor Microcystin LA.”

N-Ac-ADDA-OMe. To 31 mg (0.70 mmol) of Boc-ADDA-OMe in a flask was added2 ml of TFA. After one hour the TFA was removed under vacuum, and theresidue was concentrated three times from toluene to remove the TFA. Theresulting oil was dissolved in 2.5 ml of freshly distilled CH₂Cl₂, andthis was cooled to 0° C. 28 mg (0.28 mmol) of anhydrous triethylaminewas added to the solution, followed by 0.141 g (1.39 mmol) of freshlydistilled acetic anhydride. One hour later 5 ml of saturated NH₄Cl wasadded, and the mixture was stirred for 20 minutes at 0° C. The mixturewas partitioned between water and EtOAc, and the phases were separated.The aqueous phase was extracted twice with EtOAc. The combined organicphases were washed once each with 50% saturated NH₄Cl, 50% saturatedNaHCO₃, and brine, dried over MgSO₄, filtered, and concentrated undervacuum to give a white solid. The solid was purified via flashchromatography (1/1 EtOAc/hexanes) to give 25 mg (93%) of a white solid:R_(f) 0.23 (40:60 EtOAc:hexanes); IR (thin film) 3330, 2919, 1731, 1654,1454 cm⁻¹; ¹HNMR (500 MHz, CDCl₃) 0/00 0.99 (d, J=6.5, 3H), 1.20 (d,J=7, 3H), 1.57 (s, 3H), 2.02 (s, 3H), 2.58 (ddq, J=6, 6.5, 10 Hz, 1H),2.67 (dd J=7.5, 14 Hz, 1H), 2.78 (obscured mult., 3H), 2.79 (dd, J=5,13.5 Hz, 1H), 3.17 (ddd, J=5, 6, 7 Hz, 1H), 3.21 (s, 3H), 3.65 (s, 3H),4.71 (ddd, J=4.5,5,5.5 Hz, 1H), 5.37 (d, J=9.5 Hz, 1H), 5.42 (dd,J=15.5, 6.5 Hz, 1H), 6.18 (d, J=15.5 Hz, 1H), 6.40 (d, J=9 Hz, 1H),7.25-7.15 (m, 5H); ¹³C NMR (125 MHz, CDCl₃) 0/00 175.8, 169.5, 139.5,136.6, 136.3, 132.4, 129.5, 128.2, 126.0, 124.9, 87.1, 58.6, 3.0, 51.7,43.6, 38.4, 36.8, 23.5, 16.2, 14.9, 12.7; HRMS calculated for C₂₃H₃₄NO₄(M+H)⁺: 388.2488. Found: 388.2505.

N-Ac-ADDA-OH. To 22 mg (0.057 mmol) of the protected ADDA-derivative in2 ml THF was added 0.57 ml (0.57 mmol) of 1 M LiOH. After 22 hours themixture had clarified, and it was partitioned between hexanes and water.The phases were separated, and the aqueous phase was washed once withhexanes. The combined organic phases were back-extracted three timeswith water. The combined aqueous phases were acidified with 1 M NaHSO₄,and extracted three times with CH₂Cl₂. The combined CH₂Cl₂ phases werewashed once with brine, filtered through cotton, and concentrated togive 23 mg of 83 as an oil that was taken on without purification: R_(f)0.34 (1:49:50 HOAc:EtOAc:hexanes); IR (thin film) 3295 br, 2923, 1713,1640 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) % 0.99 (d, J=6.5, 3H), 1.25 (d, J=7,3H), 1.58 (s, 3H), 2.02 (s, 3H), 2.57 (ddq, J=6.5, 6.5, 9.5 Hz, 1H),2.65 (dd J=7.5, 14 Hz, 1H), 2.76 (par.obsc. m, 3H), 2.77 (dd, J=5, 13Hz, 1H), 3.17 (ddd, J=5, 6.5, 6.5 Hz, 1H), 3.21 (s, 3H), 4.71 (ddd, J=5,6, 10 Hz, 1H), 5.37 (d, J=9.5 Hz, 1H), 5.45 (dd, J=15.5, 6.5 Hz, 1H),6.18 (d, J=15.5 Hz, 1H), 6.37 (d, J=9.5 Hz, 1H), 7.25-7.15 (m, 5H); HRMScalculated for C_(22H) ₃₂NO₄(M+H)^(+:)374.2331, Found: 374.2325.

N-Ac-ADDA-D-Ala-OMe. To 17 mg (0.12 mmol) of D-Ala-OMe hydrochloride and14 mg (0.036 mmol) of HATU in a flask was added 9 mg (0.024 mmol) of 83in 0.6 ml DMF. The resulting solution was cooled to 0° C., and 41 mg(0.34 mmol) of collidine was added. The solution was stirred at 0° C.for 2 hours, followed by warming to room temperature and stirringovernight. The mixture was partitioned between water and EtOAc, and thephases were separated. The aqueous phase was extracted once with EtOAc.The combined organic phases were washed once each with sat. NaHCO₃,water, 1 M NaHSO₄, water, and brine, dried over MgSO₄, filtered, andconcentrated under vacuum to an off-white solid. Chromatography (80:20EtOAc:hexanes) gave 8 mg (73%) of a white solid: R_(f) 0.17 (60:40EtOAc:hexanes); IR (thin film) 3284, 3067, 2923, 1743, 1650, 1542 cm⁻¹;¹H NMR (500 MHz, CDCl₃) 0/00 0.99 (d, J=6.5, 3H), 1.23 (d, J=7, 3H),1.35 (d, J=7 Hz, 3H), 1.58 (s, 3H), 2.04 (s, 3H), 2.52 (dq, J=4, 7 Hz,1H), 2.59 (ddq, J=6.5, 7, 9.5 Hz, 1H), 2.68 (dd J=7.5, 14 Hz, 1H), 2.81(dd, J=4.5, 14 Hz, 1H), 3.19 (ddd, J=5, 7, 7 Hz, 1H), 3.22 (s, 3H), 3.75(s, 3H), 4.55 (dq, J=7,7 Hz, 1H), 4.62 (m, 1H), 5.39 (d, J=9.5 Hz, 1H),5.45 (dd, J=15.5, 6.5 Hz, 1H), 6.18 (d, J=15.5 Hz, 1H), 6.23 (d, J=7 Hz,1H), 7.05 (d, J=9 Hz, 1H), 7.27-7.17 (m, 5H); ¹³C NMR (125 MHz, CDCl₃)0/00 12.7, 15.4, 16.2, 18.4, 23.5, 36.7, 38.2, 44.4, 47.9, 52.6, 53.7,58.6, 86.9, 125.2, 125.9, 128.2, 129.4, 132.2, 136.2, 139.4, 169.9,173.2, 174.6; HRMS calculated for C₂₆H₃₉N₂O₅(M+H)⁺:459.2859,Found:459.2869.

N-Ac-ADDA-D-Ala-OH. To 5 mg (0.01 1 mmol) of N-Ac-ADDA-D-Ala-OMe in1.7r) ml of THF was added 0.10 ml (0.10 mmol) of 1 M LiOH. After 50minutes, the mixture was partitoned between ether and water, and thephases were separated. The aqueous phase was washed once with ether. Thecombined etheral phases were back-extracted three times with water, andthe combined aqueous phases were acidified to pH=3 with saturated citricacid. The aqueous phases were then extracted twice with EtOAc, and thecombined EtOAc phases were washed twice with water, once with brine,dried over MgSO₄, filtered, and concentrated in vacuo. The resultingsolid was purified by preparative reversed-phase HPLC, retention time ofproduct =15.7 minutes (70 MeOH/30 0.2% aq. TFA), to give 4 mg (85%) ofthe title compound as a white solid: R_(f) 0.36 (1 HOAc/10 MeOH/89CH₂Cl₂); IR (thin film) 3288 br, 2937, 1720, 1658,1632 cm⁻¹; ¹H NMR (500MHz, DMSO-d₆) 0/00 0.94 (d, J=7.0 Hz, 3H), 0.96 (d, J=7.0 Hz, 3H), 1.19(d, J=7.0 Hz, 3H), 1.52 (s, 3H), 1.82 (s, 3H), 2.63 (dd, J=7.0, 14.0 Hz,1H), 2.73 (dd, J=5.0, 14.0 Hz, 1H), 3.16 (s, 3H), 3.22 (ddd, J=5.5, 5.5,6.5 Hz, 1H), 4.19 (dq, J=7.0, 7.5 Hz, 1H), 4.40 (m, 1H), 5.38 (d, J=10.0Hz, 1H), 5.44 (dd, J=6.5, 16.0 Hz, 1H), 6.05 (d, J=16.0 Hz, 1H), 7.17(d, J=7.5 Hz, 3H), 7.25 (t, J=7.5 Hz, 2H), 7.60 (d, J=9.0 Hz, 1H), 8.01(d, J=7.0 Hz, 1H); FAB MS calculated for C₂₅H₃₇N₂O₅(M+H)^(+:) 445.2702.Found: 445.2695.

Coupling of Hapten to Proteins

Preparation of BSA-, cBSA-, and OVA-N-AcADDA-AlaOH.

BSA (10.6 mg), cationised BSA (cBSA) (10.0 mg), and OVA (8.3 mg) wereeach dissolved in PBS (1000 μl). Carbonyidiimidazole (19.81 mg, 0.12mmol) was dissolved in dry DMF (500 μl), and a portion of the solution(100 μI) was added to N-acetyl-ADDA-D-Ala-OH (1.0 mg, 2.2 μmol) andallowed to stand for 90 min. DMF was added (BSA, 260 μl; cBSA, 260 μl;OVA, 280 μl) to the protein solutions just prior to addition of theactivated ADDA-derivative. The solution of the activated ADDA-derivative(40 μl each to the BSA and cBSA, 20 μl to the OVA) was then added to theprotein solutions, and the reaction was allowed to proceed at 4° C. forabout 16 h. The resulting conjugates were repeatedly diluted and thenconcentrated by ultrafiltration (Filtron centrifugal ultrafiltrationtubes, 10 K cutoff) until the calculated dilution of unretained lowmolecular weight compounds was >10⁶.

Preparation of HRP-MC-YR and aminoHRP.

Horse radish peroxidase (HRP) was oxidized by the method of Hermanson.HRP (19.73 mg, Boehringer) was dissolved in PBS and cooled to 4° C.NalO₄ (36.7 mg) was dissolved in water (2 ml), and 100 μl of this wasadded to the HRP solution, which rapidly became green. The reaction washeld at 4° C. in the dark for 20 min, then the HRP was separated fromlow molecular weight material by elution with PBS through a desaltingcolumn (Bio-Rad Econo-Pac 10DG).

To half of the oxidized HRP MC-YR-cysteamine (51 μg, see below) wasadded in MeOH (50 μl). To the other half diaminoethane hydrochloride(500 mg) was added in PBS (500 μl). NaBH₃CN (16.4 mg) was dissolved inPBS (500 μl), and 100 μl of this was added to each HRP reaction (whichimmediately became crimson). After standing at 4° C. in the dark forabout 16 h, the reactions were quenched by addition of diethanolamine inPBS (50 μl of 300 μl of diethanolamine in 5 ml PBS) and allowed to standat 4° C. in the dark for 2 h. The HRP solutions were then purified bypassing through desalting columns (as above). The diaminethane conjugate(henceforth referred to as aminoHRP) and MC-YR conjugates were furtherpurified by ultrafiltration to >10 ⁴ dilution (as above).

Preparation of HRP-, aminoHRP-, and OVA-ADDA-HG.

HRP, aminoHRP, and OVA were each dissolved in PBS (1 ml). To ME-ADDA-HG(0.67 mg) was added CDl (1.16 mg) in dry DMF (100 μl), and the reactionproceeded at ambient temperature 1.5 h whereupon dry DMF (150 μl) wasadded. A portion of this solution was added to the solutions of theproteins (50 μl to aminoHRP, 100 μl to HRP and OVA). DMSO (200 μl) wasthen added to the HRP and OVA reactions to assist in solubilising thereactants, and the three reactions were maintained at 4° C. in the darkfor ca 16 h. The conjugates were then purified on the desalting columnand then further purified by repeated ultracentrifugation to >3×10⁴dilution (as above).

Preparation of MC-YR-cysteamine conjugate

The method is based on those of Kondo et al. (1992) and Sherlock et al.(1998). Cysteamine (15.6 mg) was dissolved in water (500 μl), and MC-YR(500 μg) was dissolved in 5% K₂CO₃ (500 μl). The cysteamine solution (50μl, followed by 100 μl at 30 min) was added to the MC-YR solution inportions. After about 2 h the reaction was acidified to pH 3 to 4 andapplied to a reverse-phase flash column (4×1 cm). The column was elutedsuccessively with water (10 ml), 10% MeOH (10 ml), 20% MeOH (10 ml), 30%MeOH (10 ml), 50% MeOH (2×10 ml), 70% MeOH (2×10 ml), and MeOH (3×10ml). HPLC analysis indicated the product to be in the 50% MeOH and thefirst of the 70% MeOH fractions. These fractions were combined and thesolvent removed in vacuo to yield MC-YR-cysteamine as a colourless solid(204 μg). ESI-MS mlz 1121.9 (M-H⁻); ¹H, COSY and HMBC NMR spectra wereconsistent with the desired product.

Immunization of sheep and mice with ADDA-protein conjugates

Nine sheep and nine mice were immunised with the above BSA-ADDA-,cBSAADDA- and OVA-ADDA-conjugates (three animals for each conjugate).One mg of each conjugate in a volume of 1 ml phosphate buffer saline wasadded to Freund's complete adjuvants in case of the primary injectionand homogenised to form an emulsion, and Freunds incomplete adjuvants inthe case of booster injections. The animals received a minimum of threeboosts in case of sheep, and six boosts in case of mice at approximately4-week intervals.

ELISA

Indirect ELISA using polyclonal antibody #824

ELISA plates (NUNC MaxiSorp 1 #439454, Denmark) were coated withOVA-Nacetyl-D-alanyl-ADDA conjugate (OVA-ADDA-HG3/99′) in 0.05 M sodiumbicarbonate buffer pH 9.6 (75 μl, 2.5 μg/ml) overnight at 22° C. (RT).After a wash with PBS, additional binding sites were blocked byincubation with OVA (1/% w/v, 300 μl, 1 h, 20–25° C.). Plates werewashed two times with PBS and used immediately or stored at 4° C. for upto 7 days. In the assay, sample or standard (50 μl) was added to thewells together with antiserum (50 μl) at the appropriate dilution (e.g.sheep serum #824^(26/6/00) at 1/200 000; cf. FIG. 5). After incubationat 20–25° C. for 2 h, wells were washed twice with PBS +0.05% Tween® 20(PBST) and twice with PBS. Secondary antibody, horse radish peroxitaseconjugated antispecies antibody, e.g. ICN/Cappel Anti-sheep-HRP (100 μl,1/6000), was then added to the wells and incubated for 2 h. Thereafter,wells were aspirated, washed twice with PBST and twice with PBS. TMBsubstrate solution, prepared by adding 110 μl TMB stock (10 mg/ml inDMSO) to 11 ml of sodium acetate buffer (0.1 M, pH 5.5) containing0.005% H₂O₂, was then added and incubated for 15 minutes. The reactionwas stopped by addition of H₂SO₄ (50 μl, 2 M), and the absorbance at 450nm was determined with a microplate spectrophotometer. Standards andsamples were prepared for ELISA by dilution in the following diluents:(i) methanol in PBS to a maximum methanol concentration of 10% (v/v);(ii) PBS; (iii) lake water (Lake Constance, Germany); (iv) tap water;(v) river water, Waikato River, New Zealand. All samples were analysedin at least duplicate, and over a range of dilutions.

ELISA method using antibody ADDA-#824^(26/6/00) in detail

The principle of the indirect competitive microcystin ELISA (MC-ELISA)employed is shown in FIG. 4. This method comprises the following steps:

-   -   1. Prepare antigen (OVA-ADDA-HG^(3/99)) in bicarbonate buffer,        pH 9.6 at 2.5 μg/ml (5 ml +/plate).    -   2. Coat antigen onto microtitre plate at 75 μl/well, tap to mix        and cover, incubate overnight at room temperature (RT, 22° C.).    -   3. Wash two times in PBS, aspirate.    -   4. Block plate with 1% OVA (no. A-5503 from Sigma) (300 μl for 1        hour at RT (22° C.).    -   5. Wash two times in PBS, aspirate and use immediately or add        2–300 μl PBS for storage. The plates can be stored at this stage        in PBS (at 4° C.) for up to 7 days.    -   6. Add 50 μl sample, or standard, in PBS; and 50 μl of antibody        ADDA-#824^(26/6/00) (developed in sheep) at 1/200 000 dilution        in OVA-blocker and incubate for 2 hours at RT (22° C.). Standard        curve: Primary 5000 ng/ml, then nine serial 1:8 dilutions (1+7)        in 10%MeOH/PBS.    -   7. Wash two times in PBS/Tween, two times in PBS.    -   8. Add 100 μl of secondary antibody conjugate diluted in OVA        (peroxidase conjugated rabbit-anti-sheep lgG (ICN #55814) at a        final dilution of 1/6000 and incubate for 2 hours at RT (22°        C.).    -   9. Wash two times in PBST, two times in PBS, aspirate.    -   10. Turn on plate reader—needs a 15 minute warm up before        reading at step 13.    -   11. Add 100 μl of substrate. Incubate for 15 minutes at RT (22°        C.) until colour develops.    -   12. Add 50 μl stop solution (2M H₂SO₄).    -   13. Read absorbance at 450 nm. Note that the absorbance at 655        nm can be measured prior to adding the stop solution if        required.        Direct ELISA using polvclonal antibody #825

ELISA plates (NUNC Maxisorp 1 #439454, Denmark) were coated with theappropriate antiserum (#825^(14/12/98)) in 0.05 M sodium bicarbonatebuffer pH 9.6 (50 μl, 1/20 000) overnight at 20° C. After a 2×PBS wash,additional binding sites were blocked by incubation with BSA (1% w/v,300 μl, 1 h, 20–25° C.). Plates were washed two times with PBS and usedimmediately or stored at 4° C. for up to 7 days. In the assay, sample orstandard (50 μl) was added to the wells together with the appropriatehapten-enzyme conjugate (50 μl, NH₂-ADDA-HRP^(3/99), 200ng/ml). Afterincubation at 20–25° C. for 3 hours, wells were washed twice with PBSTand twice with PBS. TMB substrate solution, prepared by adding 110 μlTMB stock (10 mg/ml DMSO) to 11 ml sodium acetate buffer (0.1 M pH 5.5)containing 0.005% H₂O₂, was then added, followed by incubation for 15minutes. The reaction was stopped by addition of H₂SO₄ (50 μl, 2 M), andthe absorbance was determined with a microplate spectrophotometer at awavelength of 450 nm. Standards and samples were prepared for ELISA bydilution in the following diluents: (i) methanol in PBS to a maximummethanol concentration of 10% (w/v); (ii) PBS; (iii) lake water (LakeConstance, Germany); (iv) tap water; (v) river water (Waikato River, NewZealand). All samples were analysed at least in duplicate and over arange of dilutions.

Direct ELISA method in detail (example 99153005).

-   -   1. Prepare antiserum (#825, developed in sheep) in bicarbonate        buffer pH 9.6 at 1/20 000 (5 ml\plate). Coat microtitre plate        with 50 μl antiserum per well, tap to mix and cover, incubate        overnight at room temperature (RT, 22° C.).    -   2. Wash 2×PBS, aspirate.    -   3. Block plate with 1% BSA (300 μl for 1 h at RT (22° C.)).    -   4. Wash 2×PBS, aspirate and use or add 200–300 μl PBS for        storage. The plates can be stored at this stage in PBS (at 4°        C.) for up to 7 days.    -   5. Add 50 μl sample, or standard in PBS, and 50 μl of        hapten-enzyme conjugate (NH₂-ADDA-HRP) 200 ng/ml in BSA-blocker        and incubate at room temperature for 3 hours at RT (22° C.).        Standard curve primary 2000 ng/ml, then 9 serial 1:6 dilutions        in PBS.    -   6. Wash 2×PBST, 2×PBS. Aspirate.    -   7. Turn on plate reader—needs a 15 minute warm up before reading        at step 10.    -   8. Add 100 μl of substrate. Incubate at RT (22° C.) for 15        minutes.    -   9. Add 50 μl stop solution (2 M H₂SO₄).    -   10. Read absorbance at 450 nm. Note that the absorbance at 655        nm can be measured prior to adding the stop solution if        required.

Results of the above-described test are illustrated in FIG. 6.

Indirect ELISA using monoclonal antibody #3G10B 10 (assay 9910n001)

ELISA plates (NUNC MaxiSorp 1 #439454, Denmark) were coated withOVAADDA-HG conjugate in 0.05 M sodium bicarbonate buffer pH 9.6 (50 μl,2.5 μg/ml) overnight at 20° C. After a wash with PBS, additional bindingsites were blocked by incubation with BSA (1/% w/v, 300 μl, 1 h, 20–25°C.). Plates were washed two times with PBS and used immediately orstored at 4° C. for up to 7 days. In the assay, sample or standard (50μl) was added to the wells together with monoclonal antibody (50 μl) atthe appropriate dilution (e.g. #3G10B10 at 1/750). After incubation at20–25° C. for 2 h, wells were washed twice with PBS+0.05%

Tween® 20 (PBST) and twice with PBS. After incubation at 20–25° C. for 2h, wells were washed twice with PBS+0.05% Tween™20 (PBST) and twice withPBS. Secondary antibody, horse radish peroxitase conjugated anti-speciesantibody, e.g. Silenus DAH anti-mouse-HRP (100 μl, 1/2000), was thenadded to the wells and incubated for 2 h. TMB substrate solution,prepared by adding 110 μl TMB stock (10 mg/ml in DMSO) to 11 ml ofsodium acetate buffer (0.1 M, pH 5.5) containing 0.005% H₂O₂, was thenadded and incubated for 15 minutes. The reaction was stopped by additionof H₂SO₄ (50 μl, 2 M), and the absorbance at 450 nm was determined witha microplate spectrophotometer. Standards were prepared for ELISA bydilution in the methanol in PBS to a maximum methanol concentration of10% (v/v). All samples were analysed at least in duplicate and over arange of dilutions.

Results of the above-described test are illustrated in FIG. 7.

ELISA method using antibody #3G10B10 in detail

The principle of the indirect competitive microcystin ELISA (MC-ELISA)employed is shown in FIG. 4. This method comprises the following steps:

-   -   1. Prepare antigen (OVA-ADDA-HG3199) in bicarbonate buffer, pH        9.6 at 2.5 μml (5 ml/plate).    -   2. Coat antigen onto microtitre plate at 50 μl/well, tap to mix        and cover, incubate overnight at room temperature (RT, 22° C.).    -   3. Wash two times in PBS, aspirate.    -   4. Block plate with 1% BSA—(300 μl for 1 hour at RT (22° C.)).    -   5. Wash two times in PBS, aspirate and use immediately or add        2–300 μl PBS for storage. The plates can be stored at this stage        in PBS (at 4° C.) for up to 7 days.    -   6. Add 50 μl sample, or standard, in PBS; and 50 μl of antibody        #3G10 B10 at 1/750 dilution in BSA-blocker and incubate for 2        hours at RT (22° C.). Standard curve: Primary 1000 ng/ml, then        nine serial 1:4 dilutions (1+3) in PBS.    -   7. Wash two times in PBS/Tween, two times in PBS.    -   8. Add 100 μl of secondary antibody conjugate diluted in OVA        (Horseradish peroxidase-conjugated rabbit-anti-mouse IgG        (Silenus DAH) at a final dilution of 1/2000 and incubate for 2        hours at RT (22 ° C.).    -   9. Wash two times in PBST, two times in PBS, aspirate.    -   10. Turn on plate reader—needs a 15 minute warm up before        reading at step 13.    -   11. Add 100 μl of substrate. Incubate for 15 minutes at RT (22°        C.) until colour develops.    -   12. Add 50 μl stop solution (2M H₂SO₄).    -   13. Read absorbance at 450 nm. Note that the absorbance at 655        nm can be measured prior to adding the stop solution if        required.        PreDaration of Buffers:        Bicarbonate Coating Buffer

Dissolve 0.85 g Na₂CO₃, (or 2.15 g Na₂CO₃ ⁻2 H₂O) and 1.47 g NaHCO₃ in500 ml distilled water, adjust pH to 9.6 (gives 0.05 M bicarbonate).

Phosphate Buffered Saline (PBS)

To prepare 10 times stock solution:

NaH₂PO₄ ⁻2 H₂O 2.897 g (or NaH₂PO₄ anhydrous 2.06 g)

Na₂HPO₄ anhydrous 11.938 g

NaCl 87.660 g

Weigh phosphates, add water to 800 ml, adjust pH to 7.4, then add salt.

Add water to 1 l and check pH (must be 7.2 to 7.6).

Dilute 1/10 for use: gives 0.01 M wrt phosphate and 0.15M NaCl.

Ref.: Mishell et al. (1980)

PBS/Tween

Suspend Tween-20 at 0.05% in PBS (0.5 ml/l);

Use for the washing steps desribed above.

OVA-Blocking Buffer

Dissolve OVA (Sigma A-5503) in PBS at 1% (2 g/200 ml).

Use for blocking plates, and as diluent for Ab and Ab″.

Secondary Antibody

Also referred to herein as Ab″, HRP-conjugate, and Second Ab. Thedilution depends on the batch used, but approximate dilutions are asfollows:

ICN HRP-Conjugated Rabbit-Anti-SHEEP-IgG #55814

Use at a working dilution of 1/3000. Stock solution is stored at 1/10 inPBS thiomersal (0.02%).

TMB Substrate

Prepare stocks of:

1) Sodium acetate buffer 0.1M, pH 5.5 (1.315 g/200 ml) (check forprecipitate before use).

2) TMB (3,3′,5,5′-tetramethylbenzidine) at 10 mg/ml DMSO; [store in thedark at RT (22° C.)].

Immediately before use:

Dissolve 110 μl TMB solution (2) in 11 ml sodium acetate buffer (1),and, add 165 H₂O₂ (prepared freshly by diluting 38 μl 30% H₂O₂(commercial strength) into 2.5 ml distilled H₂O).

ELISA Plates

96-well-plates were from NUNC (Maxisorp l plates, catalogue #439454).

Characterization of polyclonal anti-ADDA-antibody developed in sheep

The optimal concentrations of assay reagents were determined empiricallyby chequerboard titrations. Assay standard curves were calculated usingMicrosoft Excel. Cutoff values of 20 to 80% of maximum absorbance wereused in order to determine the working range. Cross-reactivity of theassay was determined against congeners of MC-LR, -RR, -YR, -LW, -LF,desmethyl-MC-LR, desmethyl-MC-RR and nodularin and calculated from theconcentration of analogue giving 50% inhibition (l₅₀) of binding to theprotein-ADDA solid phase, expressed relative to the l₅₀ for freemicrocystin-LR. The calculation of the cross-reactivity demonstratesthat for sample concentrations ranging between 0.01 and 1 ng/ml theactual toxin concentrations are underestimated in the worst case by 5%.As of a sample concentration ranging betwee 1 ng/ml and 1 μg/l, mostcongeners tested are detected with equal sensitivity, i.e. 100%cross-reactivity (cf. FIG. 5), while the concentrations of MC-RR andnodularin are slightly overestimated (<5%). This demonstrates thatmicrocystin and nodularin congeners can be detected reliably over aconcentration range which is tenfold lower than the safe limit proposedby the WHO.

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1. A compound comprising one or more polypeptides providing a binding site of a monoclonal, polyclonal or recombinant antibody or a functionally active derivative or part thereof, wherein said compound is prepared using the group of formula (I) as a hapten, and said compound is capable of specifically binding to a compound having a structure of formula (I) represented as

wherein group R¹ represents a halogen atom, -OSO₃, -OR^(′) or -NR′₂ and group R² represents hydrogen, (C₁–C₄)alkyl, (C₁–C₄)alkoxy, (C₁–C₄)acyl, (C₁–C₄)acylamino, (C₁–C₄)carboxyaminoacyl, glutamidyl, or 2-aminopropionamidyl, and wherein the groups R³ which may be the same or different are each independently selected from the group consisting of hydrogen and (C₁–C₄)alkyl, group R⁴ represents (C₁–C₄) alkoxy, the phenyl group may be substituted or unsubstituted, and further wherein the groups R^(′) represent hydrogen, substituted or unsubstituted (C₁–C₄)alkyl or (C₁–C₄)acyl.
 2. The compound according to claim 1, wherein the groups R³ each represent methyl and group R⁴ represents methoxy.
 3. A compound comprising one or more polypeptides providing a binding site of a monoclonal, polyclonal or recombinant antibody or a functionally active derivative or part thereof, wherein said compound is prepared using the group of formula (I) as a hapten, and said compound is capable of specifically binding to a compound having a structure of formula (I) represented as

wherein group R¹ represents acylamino and group R² represents (C₁–C₄)acyl; or group R¹ represents glycyl or D-alanyl and group R² represents acetyl; or group R¹ represents -NH₂ and group R² represents glutamidyl or 2-aminopropionamidyl, and wherein the groups R³ which may be the same or different are each independently selected from the group consisting of hydrogen and (C₁–C₄) alkyl, group R⁴ represents (C₁-C₄)alkoxy, the phenyl group may be substituted or unsubstituted, and further wherein the groups R¹ represent hydrogen, substituted or unsubstituted (C₁–C₄)alkyl or (C₁–C₄)acyl.
 4. The compound according to claim 3, wherein the groups R³ each represent methyl and group R⁴ represents methoxy.
 5. The compound according to claim 1 or claim 3 which is a polyclonal, monoclonal or recombinant antibody or a functionally active derivative or fragment thereof.
 6. A method for preparation of the compound according to claim 1 or claim 3, said method comprising the steps of: (a) providing a compound containing a group represented by formula (I) as defined in claim 1 or claim 3; (b) coupling the compound of step (a) to an immunogenic carrier to form a conjugate; (c) immunizing an animal with the conjugate obtained in step (b); and (d) isolating the animal's blood, blood serum and/or spleenocytes.
 7. The method according to claim 6, wherein the immunogenic carrier is a polymeric substance.
 8. The method according to claim 7, wherein the polymeric substance is selected from the group consisting of polyethyleneglycol, polypeptides, proteins, polysaccharides and plastic supports.
 9. The method according to claim 8, wherein the substance is a protein and said protein is selected from bovine serum albumin, ovalbumin, cationised bovine serum albumin or horseradish peroxidase.
 10. A diagnostic kit containing the compound according to claim 1 or claim
 3. 11. An affinity matrix containing the compound according to claim 1 or claim 3 coupled to a polymeric resin.
 12. A method for detecting a compound containing a group represented by formula (I) as defined in claim 1 or claim 3, said method comprising the steps of: (a) providing a compound according to claim 1 or claim 3; (b) mixing a second compound suspected of containing a group represented by formula (I) as defined in claim 1 or claim 3 to form a mixture; and (c) performing an assay that detects binding of the compound according to claim 1 or claim to the second compound.
 13. A method for concentrating a compound containing a group represented by formula (I) as defined in claim 1 or claim 3 from a fluid or for substantially decreasing the amount of a compound containing the group represented by formula (I) in a fluid comprising the steps of: (a) preparing the compound according to claim 1 or claim 3, (b) coupling the compound obtained in step (a) to a polymeric matrix, and (c) contacting the fluid with the polymeric matrix obtained in step (b).
 14. The method according to claim 13, wherein the fluid is hemodialysis water, drinking water or water derived from rivers, lakes or oceans. 